Alpha-fetoprotein (AFP) is the main component of embryonic blood serum of mammals, which is synthesized by embryonal liver and yolk sac during perinatal development. Immediately after birth, the level of AFP in the serum sharply decreases and its expression became undetectable in healthy adult individuals (Deutsch H. F., 1991, Adv. Canc. Res. 56, 253-312). The synthesis of AFP is renewed upon malignant development of liver tumors and germinogenic teratoblastomas and could be detectable to a lesser degree in the case of chemical and mechanical damage to the liver, accompanied by regeneration, for example, during acute viral hepatitis or cirrhosis (Mizejewsly G. J., 2002, Expert Rev. Anticancer. Ther. 2: 89-115).
Human AFP is a glycoprotein consisting of 590 amino acids and comprising about 4% of a carbohydrate component (Morinaga T., et al., 1983, Proc. Natl. Acad. Sci., U.S.A., 80, 4604-4608; Pucci P. et al., 1991, Biochemistry 30, 5061-5066). One of the main properties of AFP is the noncovalent sorption of different low-molecular chemical substances, such as polyunsaturated fatty acids, steroidal hormones, metals, retinoids, hydrophobic antibiotics and others (Aussel S. & Masseyeff R., 1994, Biochem. Biophys. Res. Commun. 119: 1122-1127; Deutsch H. F., 1994, J. Tumor Marker Oncol., 9: 11-14). In early stages of embryonic development, AFP replaces albumin as a transport vehicle for fatty acids and other low-molecular substances (Deutsch H. F., 1991, Adv. Canc. Res. 56, 253-312).
AFP molecule consists of three globular structural domains bounded by 15 interchain disulfide bonds, which significantly increases the complexity of the process of assembly of a tertiary structure of a protein (Morinaga T., et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80, 4604-4608; Pucci P., et al., 1991, Biochemistry 30, 5061-5066). Furthermore, all important structural element of an AFP molecule is the carbohydrate component, which provides correct reception and functioning of the molecule (Deutsch H. F., 1991, Adv. Canc. Res. 56, 253-312).
In addition to a polypeptide chain consisting of 590 amino acid residues, the structure of the molecule of a serum embryonic AFP or that one secreted by hepatocarcinoma cells includes one oligosaccharide group linked to asparagin according to the N-type glycosylation (Yamashita K. et al., 1993, Cancer Res. 53:2970-2975). The structure of an oligosaccharide AFP chain is heterogeneous and depends on different factors: the stage of development of hepatocarcinoma or the stage of development of the embryo. Oligosaccharides affect structural properties of an AFP molecule, could be included in the content of antigenic determinants and receptor-binding centers (Deutsch H. F., 1991, Adv. Canc. Res. 56, 253-312). As distinctive from serum AFP, recombinant AFP expressed in bacterial cells is not glycosylated, which is a characteristic distinction of the product characterized in the works of Murgita (U.S. Pat. Nos. 6,331,611; 6,627,440; 6,416,734) and, consequently, has structural and functional properties distinguishing it from a serum analog and also from the recombinant AFP expressed in yeast systems. It is known that during expression of heterologic proteins in yeasts, their glycosylation is carried out in respect to the same amino acid residues as in the serum analog, but the structure of the oligosaccharides themselves significantly differ in respect to makeup, length and branching of the chain, which also predetermines certain distinctions in the structural and functional properties of corresponding proteins (Hard K. et al., 1998, FEBS Lett. 248:111).
AFP may be selectively absorbed by cells expressing specific AFP receptors (AFPR), such as embryonic cells, stem cells, activated immune cells, cancer cells or cells transformed by certain types of retroviruses (Uriel J. et al., 1989, in Jizejewsky G. I., Jakobson H. I. (eds): Biological Properties of Alpha-Fetoprotein. Boca Raton, CRC Press, vol. 2:103-117). Normal mature cells lose the ability to absorb AFP and do not express specific AFPR. In view of this property of AFP, methods have been proposed for the therapeutic use of AFP for the purpose of targeting delivering of cytostatics and other substances, suppressing the growth of cancer cells, to a tumor (Deutsch H. F., 1994, J. Tumor Marker Oncol. 9: 11-14; Tsukada Y. et al., 1994, J. Tumor Marker Oncol. 9: 99-103).
AFP has a number of functional properties, which at present are being intensively studied. The classical concept of AFP as an analog of embryonic serum albumin, is at present supplemented by data concerning the capability of AFP to carry out the regulation of the growth, development and programmed death of cells (Mizejewslcy G. J., 2002, Expert Rev. Anticancer. Ther. 2: 89-115). In particular, it was shown that a recombinant AFP, similarly to a serum and cultural analog, is capable of suppressing the growth of estrogen-dependent tumoral and normal tissues (Bennett J. A. et al., 1997, Breast Cancer Res. Treat. 45, 169-179; Bennet J. A. et al., 1998, Clinical Cancer Research, 4, 2877-2884). Recently, it was established that the oncosuppressive activity of AFP is carried out in accordance with the mechanism of triggering apoptosis, which is characterized by typical morphological changes, the arrest of growth, by cytotoxicity and DNA fragmentation (Semenkova L. N., 1997, Tumor Biol. 18, 261-274; Dudich E. I., et al., 1998, Tumor Biol. 19, 30-40; Dudich E. I.; et al., 1999, Eur. J. Biochem. 266: 1-13; Semenkova L., et al., 2003, Eur.; J. Biochem. 70: 4388-4399).
Earlier studies showed the capability of AFP to regulate differentiation and activation of immune cells. In particular, AFP is capable to suppress immune cells activated with allo- or autoantigens and to inhibit various cytokine gene expression (Yamashita K., et al., 1993, Cancer Res. 53, 2970-2975; U.S. Pat. No. 5,965,528). On the other hand, AFP induces pronounced stimulation of the growth of immature bone marrow cells, stem cells and embryonic cells (Dudich E. I., et al., 1998, Tumor Biol. 19, 30-40; U.S. Pat. No. 6,627,440).
These properties of AFP, and also increased selectivity of absorption of AFP by cancer cells in vivo (Uriel J., et al., 1989, in Mizejewslcy G. I., Jakobson H. I., eds: Biological Properties of Alpha-Fetoprotein. Boca Raton, CRC Press. vol. 2:103-117), revealed the base for its use in medicine as a therapeutic preparation in the treatment of autoimmune (U.S. Pat. No. 5,965,528) and oncological diseases (U.S. Pat. No. 6,416,734; Mizejewslcy G. J., 2002, Expert Rev. Anticancer. Ther. 2: 89-115). Furthermore, traditionally AFP is used as an oncoembryonic marker for early diagnosis of oncological diseases and pathologies of embryonical development (Deutsch H. F., 1991, Adv. Canc. Res. 56, 253-312). However, the use of natural AFP as a drug is technologically impossible because of raw material deficiency.
Traditionally, a source for the obtainment of AFP is the blood serum of pregnant women, funic embryonal serum or ascitic fluid of cancer patients. Obviously, none of these sources are acceptable for the production of a protein substance for medical purpose because, in the first place, there is extremely limited access to the source of raw material and the content of AFP therein is low, and in the second place, there is the ever-growing risk of infection with viruses or prions.
Earlier data were published relating to the expression and purification of recombinant AFP (rAFP) in different microorganisms (Yamamoto R., et al., 1990, Life Sciences, 46:1679-1686; Nishi S., et al., 1988, J. Biochem. 104: 968-972; U.S. Pat. No. 5,206,153; U.S. Pat. No. 6,331,611). Thus, the intracellular production of human rAFP was carried out in Saccharomyces cerevisiae (Yamamoto R., et al., 1990, Life Sciences, 46:1679-1686; U.S. Pat. No. 5,206,153) and Escherichia coli (U.S. Pat. No. 6,331,611; Boismenu R., et al., 1997, Protein Expression and Purification. 10:10-26). It was shown that recombinant AFP, expressed in Escherichia coli, retains the immunoregulatory and oncosuppressive activity of the embryonic analog (Boismenu R., et al., 1997, Protein Expression and Purification. 10:10-26; Bennett J. A., et al., 1997, Breast Cancer Res. Treat. 45, 169-179). The main drawback of these expression systems is the incapability to secrete heterologic protein and the extremely low level of its production. Furthermore, the obtainment of the desired product from a biomass of recombinant strain-producers required that additional procedures of denaturation and renaturation be carried out, which resulted in a significant reduction of the yield of the product and, as a consequence, a substantial increase of its cost. Also, in the case of use of bacterial expression systems, the problem of contamination of the product with the lipopolysaccharides of the shell, which have known endotoxic activity, is also important.
The technical solution most similar to the instant invention is the strain-producer of human AFP that is described in the references (Yamamoto R., et al., 1990, Life Sciences, 46:1679-1686; U.S. Pat. No. 5,206,153). In these sources yeast strain-producer Saccharomyces cerevisiae with intracellular production of human AFP is disclosed, the amino acid sequence of which comprises an additional section corresponding to the signal peptide of rat AFP. This invention identifies the product of secretion of a yeast strain, which product has the properties of a mature human AFP and has the original sequence SEQ ID NO:4, which corresponds to the sequence of a mature human AFP. This specificity distinguishes the product described in the instant invention over the earlier disclosed (Yamamoto R., et al., 1990, Life Sciences, 46:1679-1686; U.S. Pat. No. 5,206,153). Furthermore, a drawback of this strain described in the cited references is the absence of mechanisms for intracellular assembly and secretion of AFP into a cultural liquid, which significantly raises the cost, makes the process of preparing a purified recombinant AFP in preparative amounts more complex and provides an extremely low level of production of AFP. Furthermore, the authors of the cited work (Yamamoto R., et al., 1990, Life Sciences, 46:1679-1686; U.S. Pat. No. 5,206,153) obtained a modified recombinant AFP, the sequence of which also comprises signal and linker peptide, which limits the possibility for its medical use because of modification of the structure of the protein, resulting in a change of the immunological specificity and, as a result thereof, in an increase of the risk of immunoreactive pathology with intravenous or subcutaneous administration.
In the case of heterological secretion production with yeast cells of proteins, for which the correct folding takes place with the formation of disulfide bonds (among them AFP), of importance is the level of production of yeast disulfidisomerase (Pdi) with cells of a producer (Shusta E. V., et al., 1998, Nat. Biotechnol. 16: 773-777). Furthermore, action synergic with this enzyme is provided by an increased amount of the shaperon-like yeast protein BiP (Robinson A. S., et al., 1996, J. Biol. Chem. 271: 10017-10022).
In spite of the fact that yeasts are traditionally considered to be organisms free of secreted proteinases (Chung B. H. & Park K. S., 1998, Biotechnol. Bioeng. 57:245-249), for a number of proteins, including—for HSA, their degradation in the course of culturing yeasts is shown, which is related to the presence of still unidentified proteinases associated with the cell (Chung B. H. & Park K. S., 1998, Biotechnol. Bioeng. 57:245-249; Kang H. A., et al., 2000, Appl. Microbiol. Biotechnol. 53: 575-582). All of the listed factors require that they be taken into account during the creation of a yeast producer of AFP, effectively secreted in a cultural liquid.
Taking the drawbacks of the methods existing at present for the preparation of a recombinant AFP into account, it becomes obvious that there is a need for further improvement of the technology of the systems for expression and secretion of recombinant AFP, in particular the development of new recombinant strains having the capability for higher expression of a heterological protein with the provision for intracellular assembly of a native tertiary structure and subsequent secretion of the desired product into a cultural liquid.
Thus, the requirement for the development of industrially applicable methods of preparing AFP, which in respect to its properties would be identical or similar to human serum AFP and thus would make it possible to use it in those fields where human serum AFP is traditionally used, objectively follows from the state of the art.
The achievement of the stated object is possible by the creation of a new strain of microorganisms, which could produce in a cultural medium a polypeptide identical or similar to human serum AFP in respect to its properties.